Abstract Mechanical forces play critical roles in communication between T cells and Antigen Presenting Cells (APCs). Previous studies from our research group demonstrated that T cells can sense and respond to the mechanical rigidity of an activating substrate presenting ligands to TCR/CD3 and CD28, essentially an engineered APC. This discovery led to new systems for improving production of human T cells for cellular immunotherapy. In particular, a system based on soft elastomer fibers produced more cells per round of expansion than a rigid counterpart, and also rescued expansion of cells from individuals being treated for cancer. However, these previous experiments also suggested that the underlying mechanosensing response is dependent on not only the bulk modulus of the material but also its structure at scales comparable to or even larger than that of the attached cells. The overall purpose of this exploratory / developmental project is to identify what features of this mesh platform, such as fiber diameter and/or span length influence T cell mechanosensing. This will be approached in two complementary Specific Aims. In the first, a strategic set of fiber geometries that independently vary the multiple parameters of interest will be created by further developing electrospinning and microfabrication of elastomers. Comparison of multiple T cell responses to these fibers will be used to separate and quantify the effects of each parameter. The second Specific Aim focuses on the adherent T cell, characterizing the internal assemblies that lead to force generation. Together, these studies will address the current gap in knowledge of how T cells mechanically interact with topographically complex substrates, providing both insights into how these cells recognize and attack target cells and strategies to improve the emerging cellular immunotherapy market.